US9695481B2 - Polynucleotides comprising a reporter sequence operatively linked to a regulatory element - Google Patents

Polynucleotides comprising a reporter sequence operatively linked to a regulatory element Download PDF

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US9695481B2
US9695481B2 US13/823,661 US201113823661A US9695481B2 US 9695481 B2 US9695481 B2 US 9695481B2 US 201113823661 A US201113823661 A US 201113823661A US 9695481 B2 US9695481 B2 US 9695481B2
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reporter
regulatory element
polynucleotide
genotoxic
gene
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US20130302815A1 (en
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Bob Van de Water
Harm Vrieling
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ACADEMISCH ZIEKENHUIS LEIDEN ALSO ACTING UNDER LEIDEN UNIVERSITY MEDICAL CENTER (LUMC)
Universiteit Leiden
Leids Universitair Medisch Centrum LUMC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1086—Preparation or screening of expression libraries, e.g. reporter assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/12—Animals modified by administration of exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0393—Animal model comprising a reporter system for screening tests
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/142—Toxicological screening, e.g. expression profiles which identify toxicity

Definitions

  • This invention relates to methods for detecting agents that are genotoxic or are oxidative stress-inducing, and to molecules and cell lines that may be employed in such methods.
  • DNA damage may be induced by a range of agents including ultraviolet light, X-rays, free radicals, methylating agents and other mutagenic compounds. DNA damage can also be caused indirectly either by agents that affect enzymes and proteins which interact with DNA (including polymerases and topoisomerases) or by promutagens (agents that can be metabolised to become mutagenic). Any of these agents may directly or indirectly cause damage to the DNA that comprises the genetic code of an organism and cause mutations in genes. Such agents may be collectively known as genotoxic agents.
  • Exposure to a range of agents may also directly or indirectly lead to the production of reactive oxygen species that can react with various cellular biomolecules and affect their functionality. These agents are collectively known as oxidative stress-inducing agents.
  • the oldest and widely used test is the Ames test, which is a Salmonella based test for bacterial mutagenicity.
  • the Ames test has a relatively low sensitivity and fails to establish genotoxicity of compounds that interact with cellular structures that are specific for eukaryotic cells. Further, due to the low mutation frequencies that are induced by compounds, the Ames test is not suitable as a high-throughput test system.
  • SOS/umu-assay is a Salmonella bacterial test that is based on beta-galactosidase expression under control of the umuC stress response [2].
  • a bioluminescent marker is placed under control of the SOS response [3], and the assay was recently developed to increase testing throughput [4].
  • the DELL assay is based on reversion mutations in modified Saccharomyces cerevisiae strains containing a deletion in the his3 gene [5, 6]. Reversion depends on intrachromosomal recombination and is therefore restricted to specific classes of carcinogens.
  • the Greenscreen (GS) assay uses the enhanced green fluorescent protein (eGFP) coupled to the RAD54 DNA damage repair gene [7]. Validation of the yeast Greenscreen assay showed high specificity and sensitivity in the identification of a large collection of known genotoxic compounds [8].
  • the comet assay depends on higher mobility of cells with DNA breaks, in agarose gels and is a widely used test for chromosomal damage.
  • the assay is often used in combination with a chromosomal aberration test or an in vitro micronucleus test.
  • these assays have reduced sensitivity, will only score positive when using compounds that induce DNA strand breaks and are not suitable for high throughput screening.
  • H2AX is a specific variant of the H2A histone protein family and is rapidly phosphorylated by various kinases, including ATM and ATR, involved in the DNA damage response. It was proposed that phosphorylation of H2AX ( ⁇ H2AX) could be used as a sensitive marker for detection of DNA damage [11]. Recent evaluation of the ⁇ H2AX assay shows that phosphorylation of H2AX can be used to assess genotoxicity [12].
  • the Greenscreen HC assay uses the human lymphoblastoid TK6 cells and is a validated mammalian in vitro test for genotoxicity [9, 16].
  • the assay depends on an eGFP reporter linked to the GADD45a (Growth Arrest and DNA Damage) gene.
  • GADD45a is regulated by the p53 tumor suppressor and plays an important role in cell cycle control, DNA repair mechanisms and signal transduction and thereby is required for genome maintenance [17].
  • the GreenScreen HC GADD45a assay provides a reliable and sensitive genotoxicity test that allows discrimination between genotoxic and non-genotoxic carcinogens [18].
  • the fluorescent eGFP reporter can be detected using flow cytometry thereby allowing screening in a high throughput setup.
  • the GreenScreen HC assay is solely representative for the global p53 response and provides little mechanistic information on the reactivity of genotoxic compounds.
  • mice that can serve as biomarkers for exposure to genotoxic stress, namely Bscl2 (Bernardinelli-Seip congenital lipodystrophy 2 homolog (human)), Cbr3 (carbonyl reductase 3), Ephx1 (epoxide hydrolase 1, microsomal), Nope (immunoglobulin superfamily, DCC subclass, member 4), Cdkn1a (cyclin-dependent kinase inhibitor 1A (P21)), Perp (TP53 apoptosis effector), Pltp (phospholipid transfer protein), Srxn1 (sulfiredoxin 1 homolog ( S.
  • Cgref1 cell growth regulator with EF hand domain 1
  • Ltb4r1 leukotriene B4 receptor 1
  • Btg2 B-cell translocation gene 2, anti-proliferative
  • Gpx2 glutthione peroxidise 2
  • Ltb4r2 leukotriene B4 receptor 2
  • Ddit4l DNA-damage inducible transcript 4-like
  • Fosl1 Fos-like antigen 1
  • Egr1 Early growth response 1).
  • the inventors have developed highly sensitive mouse embryonic stem cell systems that allow establishing genotoxicity at non-cytotoxic concentrations.
  • a first aspect of the invention provides a polynucleotide comprising a reporter polynucleotide operatively linked to a regulatory element of a gene selected from a group consisting of the genes Bscl2, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic or oxidative stress-inducing agent.
  • reporter sequence we include the meaning of a polynucleotide whose expression is detectable by means of a suitable assay.
  • the polynucleotide may be one whose expression can be detected directly, for instance by using RT-PCR according to standard procedures in the art and as described in Example 1.
  • the reporter sequence is not the naturally occurring polynucleotide of the gene whose regulatory element the reporter sequence is operatively linked to.
  • expression of the polynucleotide can be detected indirectly.
  • the polynucleotide may encode a reporter protein, and expression of the encoded reporter protein assessed.
  • reporter protein we include the meaning of a protein that can be detected by means of an appropriate assay. It will be appreciated that the reporter protein may be one that is directly detected (e.g. a light emitting reporter protein) or one that is indirectly detected (e.g. an enzyme that produces a detectable signal).
  • the reporter sequence is one that encodes any of DsRed fluorescent protein, horse radish peroxidise (HRP), Green Fluorescent Protein (GFP) (or an analogue or derivative thereof), luciferase, chloramphenicol acetyl transferase (CAT) or ⁇ -galactosidase.
  • HRP horse radish peroxidise
  • GFP Green Fluorescent Protein
  • CAT chloramphenicol acetyl transferase
  • ⁇ -galactosidase ⁇ -galactosidase.
  • it may encode any protein whose cellular quantity can be accurately and rapidly assessed.
  • the reporter sequence may be fatal to the cells, or alternatively may allow cells to survive under otherwise fatal conditions. Cell survival can then be measured, for example using colorimetric assays for mitochondrial activity, such as reduction of WST-1 (Boehringer).
  • WST-1 is a formosan dye that undergoes a change in absorbance on receiving electrons via succinate dehydrogenase.
  • levels of mRNA transcribed from a reporter sequence can be assayed using RT-PCR (see Example 1).
  • the specific mRNA is reverse transcribed into DNA which is then amplified such that the final DNA concentration is proportional to the initial concentration of target mRNA.
  • Levels of expression can also be determined by measuring the concentration of a reporter protein encoded by the reporter sequence.
  • Assaying protein levels in a biological sample can occur using any suitable method.
  • protein concentration can be studied by a range of antibody based methods including immunoassays, such as ELISAs and radioimmunoassays.
  • immunoassays such as ELISAs and radioimmunoassays.
  • a protein-specific monoclonal antibody can be used both as an immunoadsorbent and as an enzyme-labelled probe to detect and quantify a specific protein.
  • the amount of the protein present in the sample can be calculated by reference to the amount present in a standard preparation using a linear regression computer algorithm.
  • two distinct specific monoclonal antibodies can be used to detect the specific protein. In this assay, one of the antibodies is used as the immunoadsorbent (primary antibody) and the other as the enzyme-labelled probe (secondary antibody).
  • Suitable enzyme labels include those from the oxidase group, which catalyze the production of hydrogen peroxide by reacting with substrate. Glucose oxidase is particularly preferred as it has good stability and its substrate (glucose) is readily available. Activity of an oxidase label may be assayed by measuring the concentration of hydrogen peroxide formed by the enzyme-labeled antibody/substrate reaction.
  • other suitable labels include radioisotopes such as iodine ( 125 I, 121 I) carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium ( 99m Tc), and fluorescent labels such as fluorescein and rhodamine, and biotin.
  • the concentration of a specific protein expressed by a reporter sequence may also be detected in vivo by imaging, for example when testing an agent in an animal model.
  • the reporter sequence may encode a bio-illuminescent reporter protein, or a fluorescent reporter protein.
  • the reporter protein is a light emitting protein (e.g. fluorescent) and its concentration is measured by assessing the light emitted, for example by using a fluorometer, or flow cytometry (eg. fluorescence assisted cell sorting (FACS) analysis), or fluorescence microscopy (eg confocal immunofluorescence microscopy) and (automated) live cell imaging.
  • a fluorometer e.g. fluorescence assisted cell sorting (FACS) analysis
  • fluorescence microscopy eg confocal immunofluorescence microscopy
  • the reporter sequence is one that encodes the light emitting reporter protein Discosoma sp red fluorescent protein (DsRed), and light emitting derivatives thereof.
  • DsRed is from the reef coral Discosoma sp. and is readily detectable by virtue of its red light emission.
  • An advantage of using a red fluorescent marker compared to the commonly used GFP is that it allows analysis of compounds that show green autofluorescence.
  • Derivatives of DsRed include polypeptide analogues, mutants or fragments of DsRed which are able to emit light.
  • DsRed-Express is a rapidly maturing variant of DsRed which contains nine amino acid substitutions that enhance solubility, reduce green emission, and accelerate maturation [43].
  • DsRed-express2 has been shown to allow stable and high expression of red fluorescent proteins in mammalian cells, and so in a particularly preferred embodiment, the reporter sequence is one that encodes DsRed-Express 2.
  • the DsRed-Express2 sequence may be obtained from the pDsRed-express2.1 plasmid (e.g. obtained from Clontech), which is shown in FIG. 5A .
  • the reporter sequence is one that encodes GFP.
  • determining the expression of a reporter sequence may comprise measuring the activity of the enzyme.
  • Enzyme assays typically measure either the consumption of substrate or production of product over time. It is appreciated that a large range of methods exist for determining the concentrations of substrates and products such that many enzymes can be assayed in several different ways as is well known in the art (e.g. Bergmeyer (1974)).
  • the reporter sequences can be operatively linked to the specified regulatory element using standard molecular biology techniques.
  • reporter sequence and regulatory element are linked such that the regulatory element is able to regulate the expression of the reporter sequence with which it is associated.
  • a ‘regulatory element of a gene’ we include the meaning of a polynucleotide sequence that regulates the expression of a gene with which it is associated.
  • the regulatory element when the regulatory element is operatively linked to a reporter sequence, the regulatory element is one that regulates expression of that reporter sequence.
  • the regulatory element is also one that stimulates expression of the reporter sequence in response to a genotoxic or oxidative stress-inducing agent.
  • stimulations expression it is understood that the regulatory element may act to switch on expression of the reporter sequence (i.e. from an undetectable level) in the presence of a genotoxic or oxidative stress-inducing agent, or it may act to increase existing expression of the reporter sequence in the presence of a genotoxic or oxidative stress-inducing agent.
  • Whether or not a particular regulatory element stimulates expression of a reporter sequence in response to a genotoxic or oxidative stress-inducing agent can be assayed using routine methods, including, for example, those described above and detailed in the Examples.
  • a polynucleotide comprising a candidate regulatory element operatively linked to a reporter sequence may be transfected into a cell, and expression of the reporter sequence assessed in the presence and absence of a genotoxic or oxidative stress-inducing agent.
  • the regulatory element generally contains one or more regulatory sequence motifs that are able to bind to particular transcription factors. In this way, expression is regulated by one or more transcription factors binding to the regulatory element in the presence of a genotoxic or oxidative stress-inducing agent.
  • genes Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1 we include mouse genes Gpx2 (NC_000078; SEQ ID No: 60), Ltb4r2 (NC_000080; SEQ ID No: 61), Ddit4l (NC_000069; SEQ ID No: 62), Fosl1 (NC_000085; SEQ ID No: 63) and Egr1 (NC_000084; SEQ ID No: 64), whose polynucleotide sequences (downstream of the promoter) are listed in FIG. 4 , and whose expression has been shown to be upregulated in response to genotoxic or oxidative stress.
  • genes Bscl2, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1 we also include orthologues of the mouse genes Bscl2, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1, that are present in other species (eg human, rat, monkey, dog and horse).
  • Orthologues of mouse genes Bscl2, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1, can be readily identified by a person of skill in the art, for example using sequence comparison programmes.
  • the orthologue has a polynucleotide sequence with at least 60%, or 65%, or 70%, or 75%, or 80%, or 85% or 90% sequence identity with the coding polynucleotide sequence (eg cDNA sequence) of the corresponding mouse gene, whose sequence (downstream of the promoter) is provided in FIGS. 3 and 4 , and more preferably 95% and 99% sequence identity.
  • the regulatory element of a gene comprises the promoter of the gene since the promoter immediately upstream of the transcription initiation site is expected to contain most of the regulatory sequence motifs that bind to transcription factors.
  • the regulatory element may comprise the promoter sequence of a gene selected from a group consisting of the genes Bscl2, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1.
  • the regulatory element is conveniently a polynucleotide fragment upstream of the transcription start site of the gene.
  • the regulatory element may comprise at least a 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 base pair polynucleotide sequence immediately upstream of the transcription start site of the gene.
  • Transcription start sites of the genes Bscl2, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, and Btg2 are indicated in FIG. 1 .
  • Transcription start sites of the genes Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1 are indicated in FIG. 2 .
  • the regulatory element comprises at least a 1400 or 1500 base pair polynucleotide sequence immediately upstream of the transcription start site of the gene.
  • the regulatory element of a gene selected from a group consisting of the genes Bscl2, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, and Btg2, comprises the respective polynucleotide sequences listed in FIG. 1 (SEQ ID Nos: 1-11).
  • the regulatory element of a gene selected from a group consisting of the genes Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1 comprises the respective polynucleotide sequences listed in FIG. 2 (SEQ ID Nos: 34-38).
  • genes typically have multiple regulatory sequences which may or may not be adjacent to one another.
  • regulatory sequences of genes can reside up to megabase distances away from the transcription initiation site. While regulatory sequences for the common basic transcription factors often reside close to the transcription initiation site of a gene (eg. within a few hundred base pairs), regulatory sequences responsible for responding to particular stimuli (eg. regulating expression in response to genotoxic stimuli) may either reside within a few hundred base pairs of the transcription initiation site, or further away from it. Thus, it is understood that not all regulatory sequences of a gene may be located in the promoter directly upstream of its transcription initiation site.
  • the regulatory element corresponds to two or more regulatory sequences which act together to increase expression of a reporter sequence in the presence of a genotoxic or oxidative stress-inducing agent.
  • the regulatory element may comprise one or more portions of the gene, which portions have been joined together.
  • the regulatory element of the gene comprises sequences downstream of the promoter sequence.
  • the regulatory element may comprise polynucleotide sequences that bind transcription factors which promote gene expression following DNA damage, or may comprise a 3′ untranslated region (UTR).
  • the regulatory element comprises at least one exon of the respective gene, or at least one intron of the respective gene, or at least one exon and one intron of the respective gene.
  • the regulatory element comprises an enhancer sequence of the respective gene, which acts to enhance gene expression in response to a genotoxic or oxidative stress-inducing agent.
  • the regulatory element comprises a regulatory sequence motif that is known to bind a transcription factor which serves to increase gene expression in response to a genotoxic or oxidative stress-inducing agent.
  • the regulatory element may comprise a p53 binding motif.
  • the regulatory element of a gene comprises the whole gene, such that the gene sequence encoding the endogenous protein is operatively linked to a reporter sequence.
  • the polynucleotide sequences (downstream of the promoter) of each of the genes: Bscl2, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, and Btg2, are provided in FIG. 3 and the polynucleotide sequences of each of the genes: Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1 (downstream of the promoter) are provided in FIG. 4 .
  • the regulatory element may be a functional derivative, or a variant (eg in which one or more nucleotides have been substituted or deleted), or a portion of one of the sequences mentioned above.
  • the regulatory element may be a variant of any of the sequences listed in FIGS. 1A-K or FIG. 2A-E , having at least, for example, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with said sequences, provided that the variant is one that stimulates expression of a reporter sequence operatively linked to it in response to a genotoxic or oxidative stress-inducing agent.
  • Percent sequence identity between two polynucleotides may be determined by any suitable method known in the art, for example by using appropriate computer programs such as WU-BLAST-2 (Altschul et al., Methods in Enzymology 266:460-480 (1996)) and Blast search, MacVector and Vector NTI (eg AlignX program in Vector NTI version 11) (Invitrogen).
  • WU-BLAST-2 Altschul et al., Methods in Enzymology 266:460-480 (1996)
  • Blast search MacVector and Vector NTI (eg AlignX program in Vector NTI version 11) (Invitrogen).
  • the regulatory element may be a portion of any of the sequences listed in FIGS. 1A-K or FIGS. 2A-E or variants of said sequences, which portions have at least, for example, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the bases of said sequences, provided that the portion is one that stimulates expression of a reporter sequence operatively linked to it in response to a genotoxic or oxidative stress-inducing agent.
  • the portion is at least 50 bp or 100 bp or 200 bp or 300 bp or 400 bp or 500 bp or 600 bp or 700 bp or 800 bp or 900 bp or 1000 bp or 1100 bp or 1200 bp or 1300 bp or 1400 bp or 1500 bp in length.
  • any regulatory element of the genes can be readily identified by assessing whether or not expression of a reporter sequence operatively linked to a putative regulatory element is activated in the presence of a genotoxic or oxidative stress-inducing agent.
  • the regulatory element may also be identified by conducting mutagenesis on a promoter of a gene selected from a group consisting of the genes Bscl2, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1, when in natural association with the respective gene, and assessing whether or not expression of the gene occurs.
  • a promoter of a gene selected from a group consisting of the genes Bscl2, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1, when in natural association with the respective gene, and assessing whether or not expression of the gene occurs.
  • the regulatory elements activate expression of a reporter sequence in response to a genotoxic agent selected from any of an agent causing base damage, an agent causing bulky DNA adducts, an agent causing single-stranded DNA breaks, an agent causing double-stranded DNA breaks, an agent causing intra-strand crosslinks, or an agent causing inter-strand crosslinks.
  • a genotoxic agent selected from any of an agent causing base damage, an agent causing bulky DNA adducts, an agent causing single-stranded DNA breaks, an agent causing double-stranded DNA breaks, an agent causing intra-strand crosslinks, or an agent causing inter-strand crosslinks.
  • the genotoxic agent may be an intercalator, a topoisomerase II poison or a methylating agent or an alkylating agent. It will be appreciated that there are no genotoxic agents that selectively induce only one type of DNA damage. Hence, as well as causing base damage, the genotoxic agent may also cause
  • the regulatory element stimulates expression of a reporter sequence in response to any of cisplatin, mitomycin C, doxorubicin, etoposide, methylmethane sulphonate, and N-methylnitrosurea, or to any of the genotoxic or oxidative stress-inducing compounds mentioned in the Examples, such as a genotoxin or pro-oxidant as suggested by the European Centre for Validation of Alternative Methods (ECVAM) (see Tables 2 and 3).
  • EVAM European Centre for Validation of Alternative Methods
  • the regulatory element may stimulate expression of a reporter polynucleotide in response to oxidative stress-inducing agent such as a pro-oxidant and including any of hydrogen peroxide, t-butyl hydroperoxide, menadione and diethyl maleate.
  • oxidative stress-inducing agent such as a pro-oxidant and including any of hydrogen peroxide, t-butyl hydroperoxide, menadione and diethyl maleate.
  • the regulatory element stimulates expression of the reporter sequence in response to a non-cytotoxic concentration of a genotoxic or oxidative stress-inducing agent.
  • the regulatory element stimulates expression of the reporter sequence in response to a concentration of genotoxic or oxidative stress-inducing agent that induces apoptosis in less than 35% of a population of cells, such as less than 30%, 20% or 10% of a population of cells.
  • Methods for assessing apoptosis are well known in the art and include annexin V and caspase 3 staining assays as described in Example 1.
  • the polynucleotide may be RNA (eg mRNA) or DNA, although typically it is DNA.
  • a second aspect of the invention provides a vector comprising a polynucleotide according to the first aspect of the invention.
  • Suitable vectors are ones which propagate in and/or allow expression of the reporter sequence in prokaryotic (e.g. bacterial) or eukaryotic (e.g. mammalian) cells.
  • the vector may be a plasmid, a cosmid, a phage or a bacterial artificial chromosome (BAC).
  • the polynucleotide sequence of the vector will depend upon the nature of the intended host cell, the manner of the introduction of the polynucleotide of the first aspect of the invention into the host cell, and whether episomal maintenance or integration is desired.
  • the vector comprises at least one selectable marker such as antibiotic resistance (e.g. kanamycin or neomycin).
  • Vectors are useful to replicate the polynucleotide of the first aspect of the invention, and are also useful to transfect cells with the polynucleotide, and may also promote expression of the reporter sequence.
  • Typical prokaryotic vector plasmids are: pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories (Richmond, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540 and pRIT5 available from Pharmacia (Piscataway, N.J., USA); pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16A, pNH18A, pNH46A available from Stratagene Cloning Systems (La Jolla, Calif. 92037, USA).
  • a typical mammalian cell vector plasmid is pSVL available from Pharmacia (Piscataway, N.J., USA). This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T-antigen-producing cells, such as COS-1 cells. Another example is pcDNA3.1 (neo) (Invitrogen) for use in COS-1 or COS-7 cells.
  • An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia (Piscataway, N.J., USA). This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.
  • Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems (La Jolla, Calif. 92037, USA).
  • Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3.
  • Plasmids pRS413-416 are Yeast Centromere plasmids (YCps).
  • the vector which comprises the polynucleotide of the first aspect of the invention is pDsRed-expression2.1 plasmid (Clontech; Strack et al, “A noncytotoxic DsRed variant for whole-cell labelling” Nat Methods, 2008, 5(11): 955-7 ( FIG. 5 )).
  • Any suitable method known in the art may be used to construct vectors containing the polynucleotide of the first aspect of the invention.
  • One such method involves ligation via homopolymer tails.
  • Homopolymer polydA (or polydC) tails are added to exposed 3′ OH groups on the DNA fragment to be cloned by terminal deoxynucleotidyl transferases.
  • the fragment is then capable of annealing to the polydT (or polydG) tails added to the ends of a linearised plasmid vector. Gaps left following annealing can be filled by DNA polymerase and the free ends joined by DNA ligase.
  • Another method involves ligation via cohesive ends.
  • Compatible cohesive ends can be generated on the DNA fragment and vector by the action of suitable restriction enzymes. These ends will rapidly anneal through complementary base pairing and remaining nicks can be closed by the action of DNA ligase.
  • the regulatory element of the polynucleotide of the first aspect of the invention may be produced so as to be flanked by desired restriction enzyme sites. The regulatory element may then be cloned into a vector that contains a reporter sequence downstream of where the regulatory element is inserted.
  • a further method uses synthetic molecules called linkers and adaptors.
  • DNA fragments with blunt ends are generated by bacteriophage T4 DNA polymerase or E. coli DNA polymerase I which remove protruding 3′ termini and fill in recessed 3′ ends.
  • Synthetic linkers, pieces of blunt-ended double-stranded DNA which contain recognition sequences for defined restriction enzymes, can be ligated to blunt-ended DNA fragments by T4 DNA ligase. They are subsequently digested with appropriate restriction enzymes to create cohesive ends and ligated to an expression vector with compatible termini.
  • Adaptors are also chemically synthesised DNA fragments which contain one blunt end used for ligation but which also possess one preformed cohesive end.
  • Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, Conn., USA.
  • Generating a BAC comprising the polynucleotide of the first aspect of the invention may be done using BAC TransgeneOmics technology as described in Poser et al, 2008 Nature Methods, 5: 409-415.
  • a third aspect of the invention provides a cell comprising a polynucleotide according to the first aspect of the invention, or a vector according to the second aspect of the invention.
  • Such cells may be used to replicate the polynucleotide of the first aspect of the invention, or may be used in a genotoxic or oxidative stress-inducing screening assay, as discussed in more detail below.
  • the cell can be either prokaryotic or eukaryotic.
  • construction and amplification of the polynucleotide of the first aspect of the invention is conveniently performed in bacterial cells, whereas the use of the polynucleotide for genotoxic or oxidative stress-inducing screening is typically limited to mammalian cells.
  • Bacterial cells are preferred prokaryotic host cells and typically are a strain of E. coli such as, for example, the E. coli strains DH5 available from Bethesda Research Laboratories Inc., Bethesda, Md., USA, and RR1 available from the American Type Culture Collection (ATCC) of Rockville, Md., USA (No ATCC 31343).
  • E. coli strains DH5 available from Bethesda Research Laboratories Inc., Bethesda, Md., USA, and RR1 available from the American Type Culture Collection (ATCC) of Rockville, Md., USA (No ATCC 31343).
  • Preferred eukaryotic host cells include yeast, insect and mammalian cells or cell lines, preferably vertebrate cells or cell lines such as those from a mouse, rat, monkey or human.
  • the cells may be stem cells (e.g. embryonic stem cells) or they may be immortalised cells (e.g.
  • hTert immortalised primary human fibroblasts may be primary cells such as hepatocytes (e.g. obtained through differentiation of stem cells).
  • Yeast host cells include YPH499, YPH500 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.
  • Preferred insect cells are Sf9 cells which can be transfected with baculovirus expression vectors.
  • Cells used for expressing the reporter sequence are ideally stably transfected. However, it is appreciated that non-stable transfectants (eg when using viral expression vectors) may also be used.
  • the cell used for expressing the reporter sequence is one that has a non-compromised DNA damage response.
  • the cell may have a functional DNA repair response and so be capable of one or more of, and preferably all of, inter-strand crosslink repair, base excision repair, homologous recombination, and non-homologous end joining.
  • the cell may have operational cell cycle checkpoints, such as G1-S, intra S, G2 and mitosis checkpoints.
  • the cell may have a functional apoptotic pathway.
  • Methods of determining whether a cell has a non-compromised DNA damage response are routine in the art, and include, for example, sequence analysis of genes, and functional assays of proteins (eg following genotoxic insult), known to be involved in the DNA damage response.
  • the cell has a functional p53 protein which is required for the functionality of some cell cycle checkpoints and for the induction of apoptosis.
  • the cell may be an embryonic stem cell or a hTert immortalised primary human fibroblast.
  • mouse embryonic stem cells are particularly useful cell lines in the method of the invention.
  • the cell is a mouse stem cell, most preferably an embryonic stem cell.
  • a non-genotoxic or a non-oxidative stress-inducing agent may be converted into a genotoxic or into an oxidative stress-inducing agent, respectively, within a cell.
  • the cell is conveniently a cell that expresses metabolising enzymes, such as an hepatocyte (eg Hep G2 or an hepatocyte derived from an embryonic stem cell), or a STO (mouse embryonic fibroblast) cell, or a lung epithelial cell (eg one grown in 3D cell culture).
  • Transformation of appropriate cells with a vector is accomplished by well known methods that typically depend on the type of vector used. With regard to transformation of prokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110 and Sambrook et al (2001) Molecular Cloning, A Laboratory Manual, 3 rd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Transformation of yeast cells is described in Sherman et al (1986) Methods In Yeast Genetics, A Laboratory Manual , Cold Spring Harbor, N.Y. The method of Beggs (1978) Nature 275, 104-109 is also useful.
  • reagents useful in transfecting such cells for example calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877, USA. Electroporation may also be used as described in Example 1.
  • the present invention also contemplates a culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium.
  • the polynucleotide of the first aspect of the invention or vector of the second aspect of the invention may be incorporated into a cell of an organism in vivo, or into the cell of a tissue ex vivo.
  • the invention also includes an organism or tissue that comprises the polynucleotide of the first aspect of the invention or vector of the second aspect of the invention.
  • the invention includes a non-human transgenic mammal that comprises a cell of the third aspect of the invention.
  • the mammal is a laboratory animal, such as any of a rodent (eg mouse or rat), a primate, a dog or a cat.
  • the polynucleotide of the first aspect of the invention can be used to screen for genotoxic or oxidative stress-inducing agents in vivo or ex vivo.
  • the polynucleotide may be incorporated into a laboratory animal such as a primate, mouse or rat that can be used to screen for genotoxic or oxidative stress-inducing agents.
  • the organism or tissue may comprise more than one of the polynucleotides, vectors or cells of the invention, each one containing a regulatory element of a different gene.
  • the invention provides an organism or tissue that comprises (i) a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and (ii) one or more cells that comprise a reporter sequence operatively linked to a regulatory element of a gene, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent.
  • the organism or tissue may comprise (i) a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and (ii) one or more cells that comprise a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of a gene selected from a respective one or more of (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 of) Srxn1, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1 and Egr1.
  • a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene selected from a respective one or more of (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15
  • the cells comprising the various reporter sequences operatively linked to regulatory elements may be the same or different.
  • the organism or tissue may comprise a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and, either in the same or respectively different cells, one or more polynucleotides that comprise a reporter sequence operatively linked to a regulatory element of a gene, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent.
  • the invention provides a cell that comprises more than one polynucleotide or vector of the invention, each one containing a regulatory element of a different gene (eg Bscl2 and Srxn1 genes).
  • the different regulatory elements may be responsive to different genotoxic or oxidative stress-inducing agents.
  • the regulatory element of one gene may be responsive specifically to genotoxic agents and the regulatory element of another gene responsive specifically to oxidative stress-inducing agents.
  • Bscl2 reporters are selectively activated following exposure to genotoxic stress
  • Srxn1 reporters are selectively activated following exposure to oxidative stress
  • Btg2 reporters are activated by both genotoxic and oxidative stress.
  • the organ or tissue comprises a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Srxn1 gene. It is understood that the cells may be same.
  • the organ or tissue comprises a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Srxn1 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Btg2 gene. It is understood that the cells may be the same.
  • regulatory elements that respond in a specific way are operatively linked to different reporter sequences.
  • a regulatory element that is responsive to genotoxic agents may be linked to a first reporter sequence and a regulatory element that is responsive to oxidative stress-inducing agents may be linked to a second reporter sequence.
  • the property of the agent can readily be determined by the readout from the different reporter sequences.
  • the polynucleotide of the first aspect of the invention or vector of the second aspect of the invention may be stably integrated into the genome of a cell in vivo or ex vivo, using standard methods in the art.
  • the polynucleotide may be integrated by regular transgene methods such as cre-lox.
  • the polynucleotide may be somatically incorporated.
  • somatic incorporation of reporter constructs in rodents can be achieved through tail vein injection.
  • the invention does not provide a process for cloning a human being, nor does it provide a process for modifying the germ line identity of human beings, nor does it provide a use of a human embryo for industrial or commercial purposes, nor does it provide a process for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such a process.
  • a fourth aspect of the invention provides a method of detecting a genotoxic or an oxidative stress-inducing agent comprising subjecting a cell according to the third aspect of the invention to a test agent; and assessing the expression of the reporter sequence.
  • cells containing different regulatory elements may be subjected to a test agent, and so the invention similarly provides a method of detecting a genotoxic or oxidative stress-inducing agent comprising (a) subjecting one or more cells that comprise a reporter sequence operatively linked to a regulatory element of a gene, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent, to a test agent, and (b) assessing the expression of the one or more reporter sequences; wherein at least one cell subjected to a test agent in step (a) comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene.
  • the method may involve subjecting cells to a test agent, wherein the cells contain regulatory elements of different genes operatively linked to a reporter sequence, provided that at least one cell comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene.
  • regulatory elements it is believed that more potentially genotoxic and oxidative stress-inducing agents can be detected, and also further insight into the mode of toxicity can be gained.
  • the different regulatory elements may be operatively linked to the same or different reporter sequences.
  • the polynucleotides that comprise the different regulatory elements operatively linked to a reporter sequence may be present in the same or different cells.
  • a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene and (ii) one or more cells that comprise a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of a gene selected from a respective one or more of (e.g.
  • a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Srxn1 gene are subjected to a test agent, which cells may or may not be the same.
  • a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Srxn1 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Btg2 gene are subjected to a test agent, which cells may or may not be the same.
  • the test agent may be any physical or chemical agent in the environment to which organisms are exposed.
  • the method may be used to screen compounds, such as candidate medicaments, food additives, plasticisers or cosmetics to assess whether it is safe to expose an organism (e.g. human) to the compounds.
  • the method may be used to assess contamination of samples with genotoxic or oxidative stress-inducing agents. For instance, the presence of genotoxic or oxidative stress-inducing agents in water supplies or in industrial effluents may be assessed.
  • the method may involve assessing the expression of any one or more reporter proteins such as a DsRed fluorescent protein, horse radish peroxidise (HRP), Green Fluorescent Protein (GFP) (or an analogue or derivative thereof), luciferase, chloramphenicol acetyl transferase (CAT) or ⁇ -galactosidase, in the presence of a test agent.
  • a DsRed fluorescent protein e.g.
  • the reporter sequence may be the naturally occurring polynucleotide of the gene whose regulatory element the reporter sequence is operatively linked to.
  • the different regulatory elements may be operatively linked to the same or different reporter sequences.
  • regulatory elements that respond in a specific way (e.g. to genotoxic agent or oxidative stress-inducing agents or classes thereof or to both genotoxic and oxidative stress-inducing agents) to different reporter sequences, such that the mode of toxicity of a particular agent can be readily determined from the reporter readout.
  • the method is performed in vitro.
  • in vitro we include the meaning of cell-based assays.
  • cell-based assays we include the meaning of cell cultures in two dimensions, such as on plastic or glass culture plates, as well as cell cultures which are cultured in three dimensional matrices.
  • These matrices may be composed of natural matrix components and contain, for example, collagen, fibronectin, laminin, or matrigel, or they may be composed of artificial matrix components.
  • the method is performed by growing cells transfected with a vector that comprises a reporter sequence operatively linked to a regulatory element of a gene, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent (e.g. one or more cells according to the third aspect of the invention), incubating the cells with a genotoxic or oxidative stress-inducing agent, and assessing the expression of the one or more reporter sequences from a sample of the cells.
  • a genotoxic agent e.g. one or more cells according to the third aspect of the invention
  • a genotoxic agent e.g. regulatory elements of any of the genes Bscl2, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1,
  • the cells may be seeded on a multiwell plate, such as a 96-well plate.
  • a multiwell plate such as a 96-well plate.
  • cells containing a fluorescent reporter protein eg DsRed
  • DsRed a fluorescent reporter protein
  • the method is performed in vivo or ex vivo.
  • one or more polynucleotides that comprise a reporter sequence operatively linked to a regulatory element of a gene, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent e.g. polynucleotides of the first aspect of the invention
  • each polynucleotide comprising a different regulatory element may have been incorporated into either the same or respectively different cells of a living organism (e.g. a laboratory animal such as a mouse or rat), and the expression of the one or more reporter sequences in the organism assessed in the presence and absence of a test agent.
  • the method is performed in vivo using transgenic animals (e.g. mice) and expression of reporter sequences assessed using whole-body small animal imaging machines.
  • the one or more polynucleotides that comprise a reporter sequence operatively linked to a regulatory element of a gene, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent, each polynucleotide comprising a different regulatory element may have been incorporated into a tissue ex vivo, and the expression of the reporter sequences in the tissue assessed in the presence and absence of a test agent.
  • Performing the method ex vivo typically involves using isolated cells from transgenic animals. For example, following hydrodynamic tail injection of the polynucleotide of the first aspect of the invention into a mouse or rat, reporter activity may be assessed in liver slices ex vivo.
  • the polynucleotide of the first aspect of the invention can be used in a genotoxic or oxidative stress-inducing screening assay to detect non-genotoxic agents or non-oxidative stress-inducing agents that may be converted into genotoxic agents or oxidative stress-inducing agents in a cell.
  • a genotoxic or oxidative stress-inducing screening assay to detect non-genotoxic agents or non-oxidative stress-inducing agents that may be converted into genotoxic agents or oxidative stress-inducing agents in a cell.
  • a reporter sequence operatively linked to a regulatory element of a gene, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent are metabolically active, or that metabolising cells are added to the cells (either as feeder cells or as a co-culture), or that a metabolising cell extract (eg. liver extract such as rat S9 mix) is added to the cells.
  • a metabolising cell extract eg. liver extract such as rat S9 mix
  • Expression of the reporter sequence may be assessed at several intervals over time. For example, expression may be assessed at 8 h, 12 h, 16 h, 24 h, 30 h and 48 h after incubation with the test agent.
  • the inventors have identified 16 genes whose expression is activated in response to genotoxic or oxidative stress-inducing agents, and which therefore may be used as biomarkers for genotoxic or oxidatively induced stress. It follows that an agent may counteract genotoxic or oxidative stress by reducing or preventing the expression of such genes upon exposure to a genotoxic or oxidative stress-inducing agent.
  • the reporter sequence of the first aspect of the invention may also be used to identify agents that counteract genotoxic or oxidative stress, for example by assessing the expression of the reporter sequence in the presence of a genotoxic or oxidative stress-inducing agent and by determining what effect a test agent has on expression.
  • a fifth aspect of the invention provides a method of detecting an agent to counteract genotoxic or oxidative stress, the method comprising:
  • cells in the method may be desirable to use cells in the method that comprise a different regulatory element operatively linked to a reporter sequence, either in the same or different cells.
  • the different regulatory elements may be operatively linked to the same or different reporter sequences.
  • regulatory elements that respond in a specific way (e.g. to genotoxic agent or oxidative stress-inducing agents or classes thereof or to both genotoxic and oxidative stress-inducing agents) to different reporter sequences, such that the counteracting effect of an agent on different types of toxicity can be readily determined by the reporter readout from the reporter sequences.
  • the invention similarly provides a method of detecting an agent to counteract genotoxic or oxidative stress, the method comprising a) subjecting one or more cells that comprise a reporter sequence operatively linked to a regulatory element of a gene, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent, to a genotoxic or oxidative stress-inducing agent, and to a test agent; and
  • At least one of the cells comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene.
  • step (a) may comprise subjecting either the same or respectively different cells that comprise different regulatory element operatively linked to a reporter sequence, to a genotoxic or oxidative stress-inducing agent, and to a test agent. It is appreciated that the different regulatory elements may be operatively linked to the same or different reporter sequences.
  • a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene and (ii) one or more cells that comprise a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of a gene selected from a respective one or more of (e.g.
  • Srxn1, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1, are subjected to a genotoxic or oxidative stress-inducing agent, and to a test agent. It is understood that the cells may be the same or different.
  • a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Srxn1 gene are subjected to a genotoxic or oxidative stress-inducing agent, and to a test agent.
  • a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Srxn1 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Btg2 gene are subjected to a genotoxic or oxidative stress-inducing agent, and to a test agent.
  • an agent that ‘counteracts genotoxic or oxidative stress’ we include the meaning of an agent that prevents or reduces DNA damage caused by a genotoxic agent, or the oxidative stress caused by an oxidative stress-inducing agent.
  • the method comprises first assessing the expression of the reporter sequence in the presence of a genotoxic or oxidative stress-inducing agent, but in the absence of a test agent, and subsequently comparing the expression with the expression of the reporter sequence in the presence of a genotoxic or oxidative stress-inducing agent, and a test agent.
  • the invention includes a method of determining whether an agent (e.g. compound) can counteract genotoxic or oxidative stress, the method comprising:
  • test agent determines whether the test agent counteracts the effect of the genotoxic agent or oxidative stress-inducing agent on the expression of the reporter sequence.
  • the invention includes a method of determining whether an agent (e.g. compound) can counteract genotoxic or oxidative stress, the method comprising:
  • step (a) determining whether the test agent counteracts the effect of the genotoxic agent or oxidative stress-inducing agent on the expression of the one or more reporter sequences; wherein at least one of the cells in step (a) comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene. It is appreciated that the cells in step (a) containing different regulatory elements operatively linked to reporter sequences may be the same or different, and that the different regulatory elements may be operatively linked to the same or different reporter sequences.
  • the expression of the reporter sequence in the presence of a genotoxic or oxidative stress-inducing agent alone may already be known, in which case it is only necessary to assess the expression of the reporter sequence in the presence of both the genotoxic or oxidative stress-inducing agent, and test agent.
  • the test agent may be any compound. Examples include any of a polypeptide, a peptide, a nucleic acid, a small molecule (e.g. less than 5000 daltons), or a natural product.
  • the method of the fifth aspect of the invention may be performed in vitro, in vivo or ex vivo, in the same way as the method of the fourth aspect of the invention.
  • a sixth aspect of the invention provides a kit of parts comprising:
  • the invention similarly provides a kit of parts comprising:
  • cells in part (i) containing different regulatory elements operatively linked to reporter sequences may be the same or different.
  • the kit of parts may contain one or more cells that comprise a different regulatory element (e.g. 2, 3, 4 or 5 different regulatory elements) operatively linked to a reporter sequence.
  • the cells may the same or different. It is appreciated that the different regulatory elements may be operatively linked to the same or different reporter sequences.
  • the kit of parts comprises (i) a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and (ii) one or more cells that comprise a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of a gene selected from a respective one or more of Srxn1, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1. It is understood that the cells may be the same or different.
  • the kit of parts comprises a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Srxn1 gene.
  • the kit of parts comprises a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Srxn1 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Btg2 gene.
  • the means for detecting expression of the reporter sequence may be any suitable means that can be used to assess expression of a particular reporter sequence.
  • the means may comprise a primer or probe.
  • the means may be an agent that can be used to measure expression of the reporter protein, such as an antibody or an enzyme substrate.
  • the kit may further comprise a cell which does not contain a reporter sequence operatively linked to a regulatory element of a gene which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent (e.g.
  • the kit may further comprise a cell which does not contain a polynucleotide according to the first aspect of the invention, or a vector according to the second aspect of the invention.
  • a further aspect of the invention provides a method of determining the effect of a genotoxic agent or an oxidative stress-inducing agent, the method comprising subjecting a cell according to the third aspect of the invention to a genotoxic agent or an oxidative stress-inducing agent; and assessing the expression of the reporter sequence. It is believed that by assessing the effect of various genotoxic agents or oxidative stress-inducing agents, agents may be classified by their effect and insights gained into modes of toxicity.
  • the invention provides a method of determining the effect of a genotoxic agent or an oxidative stress-inducing agent, the method comprising the steps of (a) subjecting one or more cells that comprise a reporter sequence operatively linked to a regulatory element of a gene, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent, to a genotoxic agent or an oxidative stress-inducing agent; and (b) assessing the expression of the one or more reporter sequences, wherein at least one of the cells comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene.
  • step (a) may comprise subjecting one or more cells that comprise a different regulatory element (e.g. 2, 3, 4 or 5 different regulatory elements) operatively linked to a reporter sequence, to a genotoxic or oxidative stress-inducing agent, and to a test agent.
  • a different regulatory element e.g. 2, 3, 4 or 5 different regulatory elements
  • the cells may the same or different. It is appreciated that the different regulatory elements may be operatively linked to the same or different reporter sequences.
  • a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene and (ii) one or more cells that comprise a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of a gene selected from a respective one or more of Srxn1, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1, are subjected to a genotoxic or oxidative stress-inducing agent. It is understood that the cells of (i) and (ii) may be the same or different.
  • a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Srxn1 gene are subjected to a genotoxic or oxidative stress-inducing agent.
  • a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Srxn1 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Btg2 gene are subjected to a genotoxic or oxidative stress-inducing agent.
  • the method may be performed in vitro, in vivo, or ex vivo as described above.
  • a yet further aspect of the invention provides a method of selecting an agent (e.g. compound) that reduces expression of the reporter sequence in the polynucleotide of the first aspect of the invention, the method comprising:
  • the invention includes a method of selecting an agent that reduces expression of one or more reporter sequences, the method comprising:
  • At least one of the cells subjected to a test agent comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene.
  • step (a) may comprise subjecting cells that comprise a different regulatory element (e.g. 2, 3, 4 or 5 different regulatory elements) operatively linked to a reporter sequence, to a test agent.
  • the cells may the same or different. It is appreciated that the different regulatory elements may be operatively linked to the same or different reporter sequences.
  • a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene and (ii) one or more cells that comprise a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of a gene selected from a respective one or more of Srxn1, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1 are subjected to a test agent. It is understood that the cells may be the same.
  • a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Srxn1 gene are subjected to a test agent.
  • a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Srxn1 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Btg2 gene are subjected to a test agent.
  • the method may be performed in vitro, in vivo or ex vivo.
  • the one or more cells may be subjected to a combination of different test agents and the effect of the combination of test agents on the expression of the reporter sequences assessed.
  • suitable test agents include those listed above.
  • Suitable methods for assessing the expression of the reporter sequence are as described above. By ‘reduces expression’ it is understood that the expression may be switched off (i.e. to an undetectable level), or that existing expression may be decreased.
  • the method of this aspect of the invention may be performed with or without first subjecting the one or more cells to a genotoxic or oxidative stress-inducing agent, depending upon the background level of expression of the one or more reporter sequences in the absence of a genotoxic or oxidative stress-inducing agent.
  • the background expression is very low for example, it may be desirable to first subject the one or more cells to a genotoxic or oxidative stress-inducing agent so that a baseline level expression of the one or more reporter sequences can be established to which the expression of the one or more reporter sequences in the presence of a genotoxic or oxidative stress-inducing agent can be compared.
  • a baseline level expression of the one or more reporter sequences can be established to which the expression of the one or more reporter sequences in the presence of a genotoxic or oxidative stress-inducing agent can be compared.
  • this may not be necessary if the baseline expression of the one or more reporter sequences in the presence of a genotoxic or oxidative stress-inducing agent is known.
  • the background expression of the one or more reporter sequences may be used as the baseline expression to which expression in the presence of a genotoxic or oxidative stress-inducing agent is compared to, in which case it is not necessary to first subject the cells to a genotoxic or oxidative stress-inducing agent.
  • the method is performed on isolated cells.
  • the invention includes a method of selecting an agent or a combination of agents that reduce expression of the reporter sequence in the polynucleotide of the first aspect of the invention, the method comprising:
  • the invention includes a method of selecting an agent or a combination of agents that reduce expression of one or more reporter sequences, the method comprising:
  • a) culturing one or more cells that comprise a reporter sequence operatively linked to a regulatory element of a gene, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent, in a suitable medium;
  • test agent selecting a test agent or combination of test agents that reduces expression of the one or more reporter sequences; wherein at least one of the cells comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene.
  • Preferences for the cell and the genotoxic or oxidative stress-inducing agent include those listed above.
  • FIG. 1 Polynucleotide sequences of PCR fragments containing regulatory elements of genes (A) Bscl2, SEQ ID No: 1; (B) Ephx1, SEQ ID No: 2; (C) Nope, SEQ ID No: 3; (D) Cdkn1a, SEQ ID No: 4; (E) Perp, SEQ ID No: 5; (F) Pltp, SEQ ID No: 6; (G) Srxn1, SEQ ID No: 7; (H) Cgref1, SEQ ID No: 8; (I) Ltb4r1, SEQ ID No: 9; (J) Cbr3, SEQ ID No: 10; (K) Btg2, SEQ ID No: 11. Forward and reverse primers are indicated below the sequence of each PCR fragment. Transcription start sites are indicated in a box in each sequence.
  • FIG. 2 Polynucleotide sequences of PCR fragments containing regulatory elements of genes (A) Gpx2, SEQ ID No: 34; (B) Ltb4r2, SEQ ID No: 35; (C) Ddit4l, SEQ ID No: 36; (D) Fosl1, SEQ ID No: 37; (E) Egr1, SEQ ID No: 38. Forward and reverse primers are indicated below the sequence of each PCR fragment. Transcription start sites are indicated in a box in each sequence.
  • FIG. 3 Polynucleotide sequences of genes (downstream of promoter).
  • FIG. 4 Polynucleotide sequences of genes (downstream of promoter).
  • FIG. 5 Cloning of promoter region by cloning a ⁇ 1500 bp fragment upstream of transcription start site that is believed to contain most of the regulatory promoter sequences. Also shown is a diagram of the pDsRed-Express 2.1 vector.
  • FIG. 6 Compound class specificity of Ephx1, Btg2, Perp and Cbr3 genes. Changes in gene expression of the endogenous biomarker genes were established by quantitative RT-PCR following a 16 hrs treatment of ES cells with indicated compounds. Depicted values are the average of tree independent experiments and error bars represent the standard deviation.
  • FIG. 7 Sensitivity of mouse ES cells to different genotoxic agents was established by determining the apoptotic response after exposure.
  • A Apoptosis as percentage of subG1 cells using flow cytometry and
  • B by using a Caspase3 activity assay. Error bars indicate the standard deviation of three independent experiments.
  • C Percentage of apoptotic cells by Annexin-V staining at different times after exposure to increasing concentrations of Cisplatin.
  • FIG. 8 DsRed reporter cell lines for genotoxicity and oxidative stress assessment.
  • A Flow cytometry analysis of DsRed expression in monoclonal mouse ES cell lines containing a DsRed fluorescent reporter driven by a putative biomarker gene promoter. As controls a promoterless and CMV-driven DsRed reporter cell line were used. Cell lines were exposed to 10 ⁇ M Cisplatin (Ephx1, Btg2, Perp, CMV, Promotorless) or 250 ⁇ M DEM (Cbr3) for 16 hrs.
  • B Clonal survival of wild type mouse ES cells and the DsRed reporter cell lines after treatment with Cisplatin and Etoposide. Shown data is the average of three independent experiments. Error bars represent the standard deviation.
  • FIG. 9 Expression kinetics of the Ephx1-DsRed reporter mimics the expression changes of the endogenous gene.
  • A Expression levels of the DsRed reporter and the endogenous Ephx1 gene as determined by quantitative RT-PCR.
  • B Ratio between DsRed and Ephx1 expression. Background expression of Ephx1-DsRed is higher than that of the endogenous Ephx1 gene, while the response to CisPt is comparable. Shown data are the average of four experiments.
  • FIG. 10 Differential response of the DsRed reporter cell lines to genotoxic and oxidative stress-inducing agents.
  • A DsRed reporter cell lines were exposed to 0.5 ⁇ M Etoposide or 5 ⁇ M Cisplatin and total DsRed fluorescence was determined using flow cytometry after different times of exposure.
  • B DsRed reporter cell lines were exposed to increasing concentrations of the DNA damage-inducing agents Cisplatin, Etoposide, MMS and the oxidative stress-inducer DEM. The increase in total DsRed fluorescence was determined after 30 hrs. of exposure using flow cytometry.
  • C Ratio of fold changes in DsRed expression of Btg2 vs.
  • Cbr3 DsRed reporter cell lines after exposure to different agents. Exposure to the DNA damaging agents (Cisplatin, Etoposide) resulted in a ratio of approximately 1.2. while induction of oxidative stress (DEM, MMS) resulted in a ratio of approximately 0.8. All data shown are the average of four independent experiments. Error bars indicate the standard error of the mean.
  • FIG. 11 Compound class specificity of the reporter cell lines.
  • B Fold change ratio between Btg2-DsRed and Cbr3-DsRed after exposure to the DNA damaging agents doxorubicin and mitomycin C (MMC) and the pro-oxidant CuSO 4 .
  • MMC DNA damaging agents doxorubicin and mitomycin C
  • FIG. 12 DsRed reporters are not activated by general cytotoxic stress.
  • A DsRed reporter cell lines were exposed to increasing concentrations of Cisplatin (CisPt), Wyeth-14,643 and Cyclosporin-A (CsA) and cell viability was determined by Alamar blue staining.
  • B DsRed reporter cell lines were exposed to increasing concentration of CisPt, CsA, Wyeth-14,643 and DES. Total DsRed fluorescence was determined 24, 30 and 48 hrs. after exposure using flow cytometry. Depicted values are the average of three independent experiments and error bars indicated the standard error of the mean.
  • FIG. 13 Differential response of the GFP reporter cell lines generated by BAC TransgeneOmics to genotoxic and oxidative stress-inducing agents.
  • GFP reporter cell lines were exposed to increasing concentrations of the DNA damage-inducing agents Cisplatin, Doxorubicin, MMC, and the oxidative stress-inducer DEM and CuSO 4 .
  • the increase in total GFP fluorescence was determined after 30 hrs of exposure using flow cytometry. All data shown are the average of 3 independent experiments. Error bars indicate the standard deviation.
  • FIG. 14 DsRed reporter cell lines for genotoxicity and oxidative stress assessment. Flow cytometry analysis of DsRed expression in monoclonal mouse ES cell lines containing a DsRed fluorescent reporter driven by five different putative biomarker gene promoters. Cell lines were exposed to 10 ⁇ M Cisplatin for 16 hrs.
  • FIG. 15 Genes being upregulated upon exposure to different genotoxic agents.
  • A Genes that showed the highest change in expression upon exposure (+) compared to untreated controls ( ⁇ ), were grouped in three gene sets based on fold change and p-value.
  • Gene set A contains genes that are induced by DNA damaging agents
  • gene set B contains genes that show specificity for oxidative stress
  • Gene set D contains genes that are activated by compounds that induce DNA damage, oxidative stress and the cell cycle inhibitor flavopiridol.
  • Responsive genes show a dose dependent increase in gene expression following exposure to the compounds tested. Data in the heatmaps is the average of three independent treatments and array hybridizations. Cells were exposed to low (L), medium (M) and high (H) concentrations of compounds that induce >10%, 10-30% or 30-50% apoptosis respectively.
  • FIG. 16 GFP-based mES reporter cells for genotoxicity and oxidative stress.
  • A Two putative biomarker genes, selected after genome-wide transcription profiling of mES cells, show a selective response to DNA damaging agents (Bscl2) or pro-oxidants (Srxn1). The red intensity in the heatmap represents for every treatment the expression level under non-treated and treated conditions.
  • B Selected genes were fused to a GFP fluorescent reporter using BAC transgenomics.
  • C Fluorescence microscopy analysis of Bscl2-GFP and Srxn1-GFP reporter cells following exposure to cisplatin (CisPt) or DEM.
  • FIG. 17 The Bscl2-GFP and Srxn1-GFP mES reporter cells show specificity for genotoxic compounds or pro-oxidants respectively.
  • A GFP reporter cells were exposed to CisPt and DEM and the induction in total GFP fluorescence of intact cells was determined by flow cytometry.
  • B Toxicity of CisPt and DEM is comparable in the reporter cell lines. Survival of the Bscl2-GFP and Srxn1-GFP reporter cells was determined as the fraction of intact cells after 24 h treatment by flow cytometry.
  • C Specific activation of the Bscl2-GFP reporter by the DNA damaging agents mitomycin C (MMC), doxorubicin and etoposide and of Srxn1-GFP after exposure to the oxidative stress-inducing agents sodium arsenite (NaAsO2), methyl methanesulphonate (MMS) and cadmium chloride (CdCl2).
  • (D) GFP reporter cells were exposed to the DNA damaging agents CisPt, etoposide, doxorubicin and mitomycin C (MMC) or to the oxidative stress-inducing agents DEM, sodium arsenite (NaAsO 2 ), cadmium chloride (CdCl 2 ) and methyl methanesulphonate (MMS) for 24 h and the induction in total GFP fluorescence of intact cells was determined by flow cytometry.
  • E Kinetics of Bscl2- and Srxn1-GFP reporter induction upon exposure to genotoxins or pro-oxidants. Bscl2-GFP and Srxn1-GFP reporter cells were exposed to 5 ⁇ M CisPt or 100 ⁇ M DEM and GFP expression was determined by live cell imaging up to 24 h exposure.
  • FIG. 18 Activation of the Srxn1-GFP reporter for oxidative stress depends on ROS production.
  • Bscl2-GFP and Srxn1-GFP mES reporter cells were treated with different concentration of the pro-oxidant DEM. The cells were simultaneously incubated with increasing concentrations of the ROS-scavenger n-acetyl cysteine (NAC).
  • NAC ROS-scavenger n-acetyl cysteine
  • Srxn1-GFP reporter cells were exposed to the pro-oxidants DEM, NaAsO2, CuSO4 and CdCl2. Increasing concentrations of NAC were added to the cells to reduce ROS levels.
  • FIG. 19 Activation of the Bscl2-GFP DNA damage reporter is associated with inhibition of DNA replication.
  • A Bscl2-GFP and Srxn1-GFP mES reporter cells were treated with increasing concentrations of hydroxyurea and aphidicolin.
  • B Cell viability of Bscl2-GFP and Srxn1-GFP reporter cell after exposure to hydroxyurea and aphidicolin.
  • FIG. 20 Activation of the Bscl2-GFP reporter depends on the ATR DNA damage signaling pathway.
  • the Bscl2-GFP and Srxn1-GFP mES reporter cell lines were treated with the DNA damaging agent CisPt or the DNA replication inhibitor aphidicolin in the presence of specific inhibitors for ATM (ku55933), ATR (schisandrin B) or Chk1/Chk2 signaling (UCN01).
  • FIG. 21 Activation of the Bscl2-GFP DNA damage reporter is independent of p53 and expression of Srxn1-GFP oxidative stress reporter is controlled by the Nrf2 antioxidant pathway. siRNA knockdown of p53 and Nrf2 in the Bscl2-GFP and Srxn1-GFP reporter cells followed by exposure to the genotoxic compounds CisPt or etoposide and to the pro-oxidants DEM, MMS and NaAsO2.
  • FIG. 22 Sensitivity and specificity of the GFP reporter assays using ECVAM class 1 compounds. Bscl2-GFP and Srxn1-GFP reporter cells were exposed to increasing concentrations of ECVAM-recommended carcinogens that should be positive in an in vitro genotoxicity assay. Induction of the GFP reporters was determined after 24 h exposure by flow cytometry.
  • FIG. 23 Sensitivity and specificity of the GFP reporter assays using ECVAM class 2 compounds. Bscl2-GFP and Srxn1-GFP reporter cells were exposed to increasing concentrations of ECVAM-recommended non-carcinogens that should be negative in an in vitro genotoxicity assay. Induction of the GFP reporters was determined after 24 h exposure by flow cytometry.
  • FIG. 24 Sensitivity and specificity of the GFP reporter assays using ECVAM class 3 compounds. Bscl2-GFP and Srxn1-GFP reporter cells were exposed to increasing concentrations of ECVAM-recommended compounds that are non-carcinogens or non-genotoxic carcinogens and which scored positive in one in vitrolin vivo genotoxicity test. Induction of the GFP reporters was determined after 24 h exposure by flow cytometry.
  • FIG. 25 Sensitivity and specificity of the GFP reporter assays using additional (geno)toxic compounds.
  • Bscl2-GFP and Srxn1-GFP reporter cells were exposed to increasing concentrations of (geno)toxic compounds with different reactive properties. These compounds were not specifically recommended by ECVAM for validation of in vitro genotoxicity testing. Induction of the GFP reporters was determined after 24 h exposure by flow cytometry.
  • FIG. 26 Bscl2-GFP reporter induction by progenotoxins that require metabolic activation.
  • A Bscl2-GFP reporter cells were treated with increasing concentrations of aflatoxin B1 (AFB1), benzo [a] pyrene (B[a]P), cyclophosphamide and dimethylbenz(a)anthracene (DMBA) either in the presence of S9 rat liver extract plus the required cofactors or in the presence of the cofactors only. After 3 h incubation, S9 mix and genotoxins were removed by washing and culturing of cells was continued for 24 h in regular culture medium. GFP reporter activity was established after 24 h by flow cytometry.
  • B Relative cell survival was determined by the fraction of intact cells following exposure by flow cytometry.
  • FIG. 27 Activation of the Srxn1-GFP reporter depends on ROS production and is controlled by the Nrf2 pathway.
  • Bscl2-GFP and Srxn1-GFP mES reporter cells were treated with different concentration of the pro-oxidants DEM, CuSO 4 , NaAsO 2 , CdCl 2 or MMS and simultaneously incubated with increasing concentrations of the ROS-scavenger N-acetyl cysteine (NAC). GFP reporter induction was determined after 24 h incubation by flow cytometry.
  • B Western blot analysis of Bscl2-GFP and Srxn1-GFP reporter cells that had been transfected with siRNAs against Nrf2.
  • Nrf2 protein level was determined 4 days after transfection. Hprt protein level was used as loading control.
  • C Bscl2-GFP and Srxn1-GFP reporter cells were exposed to 10 ⁇ M CisPt, 1.5 ⁇ M etoposide, 150 ⁇ M DEM, 0.5 mM MMS or 10 ⁇ M NaAsO 2 after knockdown of Nrf2 by siRNA transfection. GFP reporter activation was determined after 24 h exposure by flow cytometry.
  • FIG. 28 Activation of the Bscl2-GFP DNA damage reporter is associated with inhibition of DNA replication.
  • A Bscl2-GFP and Srxn1-GFP mES reporter cells were treated with increasing concentrations of hydroxyurea and aphidicolin. GFP reporter activation was determined after 24 h exposure by flow cytometry. Survival of Bscl2-GFP and Srxn1-GFP reporter cell after exposure to hydroxyurea and aphidicolin was determined by the fraction of intact cells by flow cytometry.
  • B Western blot analysis of cells that were exposed to 10 ⁇ M CisPt or 1.5 ⁇ M Aph in the presence of ATR (schisandrin B) or ATM (ku55933) inhibitors.
  • FIG. 29 Activation of the Bscl2-GFP and Srxn1-GFP reporters is independent of p53.
  • A Western blot analysis of p53 expression in the Bscl2-GFP and Srxn1-GFP reporter cells after transfection with siRNAs against p53.
  • B Induction of the Bscl2-GFP and Srxn1-GFP reporters upon exposure to 10 ⁇ M CisPt, 15 ⁇ M etoposide, 150 ⁇ M DEM, 0.5 mM MMS or 10 ⁇ M NaAsO 2 after p53 knockdown by siRNA transfection.
  • C Induction of the Btg2-GFP reporter by several (geno)toxic compounds after transfection with siRNAs against p53, Nrf2 or a scrambled siRNA pool as control.
  • Mouse embryonic stem (ES) cells are undifferentiated pluripotent cells that have the unique capability to divide unlimited. ES cells have an intact DNA damage response including the p53 pathway [20, 21] and are highly sensitive to various DNA damaging agents [22]. Therefore, mouse ES cells are highly suitable to use as an in vitro mammalian cell based assay for genotoxicity testing.
  • C57/Bl6 B4418 wild type ES cells were cultured as described previously [23]. Prior to exposure, cells were cultured on gelatin-coated plates in the absence of MEF feeders. Sub-confluent ES cells were exposed to different concentrations of genotoxic and non-genotoxic agents for 8 or 24 hours.
  • Cisplatin 10 ⁇ M
  • mitomycin C MMC: 0.1-0.5-1.5 ⁇ M
  • N-methylnitrosurea MNU: 0.1-0.5-2 ⁇ M
  • methyl methanesulfonate MMS: 0.1-0.2-0.5 mM
  • etoposide Etop: 0.1-0.5-2 ⁇ M
  • doxorubicin Dox: 0.01-0.05-0.2 ⁇ M
  • four pro-oxidant agents hydrogen peroxide (H 2 O 2 : 25-50-200 ⁇ M)
  • t-butyl hydroperoxide t-BHP: 25-50-100 ⁇ M
  • menadione MEN: 25-50-100 ⁇ M
  • DEM diethyl maleate
  • DES diethylstilbestrol
  • DES 0.5-2.5-10 ⁇ M
  • Wyeth14,643 1-50-250 ⁇ M
  • Cyclosporine-A CsA:
  • DsRed expression in the reporter cell lines was determined using flow cytometry (BD FACSCanto II). Cells were seeded on gelatin-coated 96 wells plates 24 prior to exposure and subsequently exposed to various genotoxic agents. Cells were washed with PBS, trypsinized and resuspended into PBS+2% serum for FACS analysis.
  • DsRed reporters were compared to expression of the endogenous genes using quantitative real time (qRT-PCR).
  • Cells were exposed to various genotoxic agents and total RNA was isolated after 6, 16 and 24 hours using the RNeasy mini kit (Qiagen).
  • cDNA was synthesized using oligo(dT) 12-18 primers and SuperscriptIII reverse transcriptase (invitrogen) according to the manufacturer's instructions.
  • Expression of DsRed and Ephx1 was determined using FastStart SYBR Green Master (Rox) QPCR mix on a 7900HT Fast Real-Time PCR System (Applied Biosystems). Relative expression was normalized using expression of the YWHAD and HPRT genes. See Table 1 for primer sequences.
  • DsRed reporter cell lines were seeded on gelatin-coated 6 well plates and treated as described above. After 24 hr., adherent cells were trypsinized and combined with the detached cells in the culture medium. Cell pellets were washed with PBS and resuspended in 0.5 ml Annexin V staining buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 5 mM KCL, 1.8 mM CaCl 2 and 1 mM MgCl 2 ) containing freshly added 1 ul/ml FITC-conjugated AnnexinV (Home made). Cells were incubated for 15 min. at room temp in the dark and analyzed by flow cytometry.
  • Annexin V staining buffer 10 mM HEPES pH 7.4, 150 mM NaCl, 5 mM KCL, 1.8 mM CaCl 2 and 1 mM MgCl 2
  • apoptosis was determined using a Ac-DEVD-AMC-based Caspase3 activity detection assay as described in Kruse et al (Mutat Res, 2007. 617(1-2): p. 58-70).
  • Ephx1, Cbr3, Btg2 and Perp Genes are Increased Upon Exposure to Genotoxic and Oxidative Stree Inducing Agents
  • Ephx1 of which expression in induced upon exposure to DNA damaging agents, is an epoxide hydrolase that is located at the membrane of the endoplasmic reticulum where it is important in the biotransformation of aromatic compounds [27].
  • the oxidative stress-induced Cbr3 gene encodes an NADPH-dependent carbonyl reductase that is involved in the reduction of a large number of biological and pharmacological compounds [28].
  • Btg2 and Perp genes respond to a broader spectrum of DNA damage and stress-inducing compounds and expression of both genes has been suggested to depend on the p53 tumor suppressor gene [29, 30].
  • Btg2 is involved in regulation of the G1 to S phase transition of the cell cycle, while Perp is a membrane protein involved in cell-cell adhesion [31-33].
  • FIG. 7A-B shows the results for Etoposide, doxorubicin, MNU and MMC (see Table 2 for a complete list of compounds used).
  • FIG. 7C we determined the level of apoptosis in time after exposure to different concentrations of CisPt by Annexin-V staining.
  • a 1500 bp DNA fragment upstream of the transcription start site of each of mouse genes Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, Bscl2, and Btg2 was fused to a recently described highly stable DsRed fluorescent protein reporter gene [34].
  • DsRed-express2 reporter [34] were cloned upstream of the DsRed-express2 reporter [34] and transfected into mouse ES cells.
  • control cell lines were generated containing either a promoterless or a CMV promoter driven DsRed reporter gene. Multiple stable clones for each construct were analyzed for DsRed expression upon exposure to genotoxic or oxidative stress-inducing agents using flow cytometry ( FIG. 8A and data not shown). For every biomarker gene a DsRed reporter cell line was selected based on low background DsRed expression and a strong increase in DsRed expression after exposure to different genotoxic compounds ( FIG. 8A ).
  • Promoterless DsRed cells did not show any DsRed expression irrespective of exposure to genotoxic agents, while the CMV-driven reporter displayed constitutive high DsRed expression when untreated but appeared to be somewhat responsive to genotoxic treatment, in agreement with previous reports [35, 36].
  • DsRed reporter cell lines were grown in 96-wells microplates, exposed to these two compounds and DsRed fluorescence was monitored using high-throughput flow cytometry at various time points.
  • both genotoxic agents induced a strong, up to 12-fold increase in total DsRed fluorescence ( FIG. 10A ).
  • DsRed expression gradually increased in time, even at 48 hours after treatment, but specifically at higher genotoxicant concentrations, high levels of apoptotic cells negatively influenced the reliability of the assay.
  • DsRed expression was determined at 30 hours after treatment using flow cytometry. In all reporter cell lines a clear induction of DsRed expression upon exposure to the different agents could be observed ( FIG. 10B ). However, the extent of induction was clearly different for some of the reporter cell lines. Btg2, Ephx1 and Perp were relatively more responsive to DNA damaging agents, while Cbr3 responded more strongly to the oxidative stress inducer DEM.
  • reporter cell lines were selectively reactive for genotoxic carcinogens or oxidative stress.
  • CsA immunosuppressant Cyclosporin A
  • DES diethylstilbestrol
  • genotoxicity test systems have been described previously that rely in fluorescent or luminescent reporter activation in response to cellular exposure to DNA damaging agents.
  • the bacterial Vitotox screen depends on a luciferase gene that is under control of the bacterial DNA damage SOS signaling system and the yeast Radarscreen is based on a ⁇ -galactosidase reporter that is controlled by the promoter of the Rad54 DNA repair gene.
  • Validation of these genotoxicity assays using the ECVAM compound list indicated that both systems show a low number of false positives and false negatives [42]. Both assays showed a strong correlation (80-90%) with the Ames mutagenicity and in vitro clastogenicity tests.
  • the mammalian Greenscreen HC assay that depends on expression of a GFP-tagged GADD45a gene that is activated by the p53-dependent DNA damage response. Also validation of this genotoxicity test system indicated a low percentage of false positives and negatives and a good correlation with mutation induction [18].
  • the sensitivity of the bacterial Vitotox, yeast RadarScreen and the mammalian Greenscreen HC assays is relatively low compared to our mES cell baser reporter system.
  • stem cells as a basis for a (geno)toxicity test system contributes to the sensitivity of the assay.
  • mES cells are proficient in all the major DNA damage response pathways, including the p53-dependent signalling pathway.
  • the use of ES cells as reporter system allows detection of genotoxicity of compounds at 10 to 100-fold lower concentrations compared to the Vitotox and Greenscreen HC assays ( FIG. 10 and [16, 42]).
  • concentration used for testing might not be a relevant issue, it becomes important when compounds are poorly soluble in water or show autofluorescence.
  • our DsRed reporter cell lines show increased DsRed expression upon exposure to various genotoxic agents already low cytotoxic concentrations ( FIG. 10 ).
  • the DsRed proteins will accumulate in the cells which will further increase the sensitivity of the cell system.
  • FIG. 13 demonstrates activated expression of GFP in reporter cell lines generated by BAC TransgeneOmics.
  • genes Srxn1, Bscl2 and Btg2 were fused to GFP via BAC recombineering.
  • GFP expression in response to the genotoxic agents Cisplatin, Doxorubicin and MMC, and the oxidative stress-inducing agents DEM and CuSO 4 was increased. The results confirm the use of these genes as biomarkers for genotoxic and oxidative stress-inducing agents.
  • FIG. 14 demonstrates activated expression of DsRed in mouse ES cells driven by the promoter regions of each of Ltb4ra, Cgref1, Cdkn1a, Pltp, and Nope. Reporter cell lines were generated as described in Example 1. The results confirm the use of these genes as biomarkers for genotoxic and oxidative stress-inducing agents.
  • mice embryonic stem (ES) cells We have identified genes that could serve as potential biomarkers for exposure to genotoxic stress by exposing mouse embryonic stem (ES) cells to various genotoxic compounds and changes in gene expression were determined.
  • ES mouse embryonic stem
  • RNA sample labeling and hybridization on Affymetrix arrays was performed according to the manufacturer's protocols (Affymetrix). The data were analyzed using Rosetta Resolver (Rosetta Biosoftware, Seattle, Wash., USA). Upon importing the Affymetrix Genechip data (CEL files), data pre-processing including background correction and normalization was performed.
  • ES cells Five genes have been identified that can serve as biomarkers for genotoxicity or oxidative stress.
  • ES mouse embryonic
  • Regulatory elements of the selected genes are fused to a DsRed reporter gene and stably integrated into mouse ES cells.
  • the fluorescent DsRed markers provide an easy and sensitive tool to study the DNA damage response in mammalian stem cells.
  • a panel of highly sensitive mouse ES cells that allow assessment of genotoxicity is developed.
  • the assay is based on expression of a DsRed fluorescent marker gene under control of different promoters that are specifically activated upon exposure to DNA damaging agents. Genotoxicity can be tested at non-cytotoxic concentrations and detection of DsRed fluorescence using flow cytometry allows usage of these cell lines as a high throughput assay.
  • the cell lines are generated, DsRed expression detected, and apoptosis assayed by, for example, methods described in Example 1.
  • Sensitivity of the reporter cell lines for detection of genotoxic stress is assessed by exposing the cells to increasing concentrations of the genotoxic agents cisplatin, etoposide, the methylating agent methylmethane sulphonate (MMS) and the indirect oxidative stress inducer diethyl malonate (DEM). At different times after treatment DsRed expression is determined using flow cytometry. Used concentrations of the different compounds is based on the level of apoptosis induction in wild type ES cells ( FIG. 7 ).
  • CsA cyclosporine A
  • DES diethylstilbestrol
  • Apoptosis can be easily determined by annexin V or caspase 3 staining assays, for example as described above.
  • DsRed expression in the reporter cells is compared to apoptosis induction after exposure to increasing doses of genotoxic agents.
  • the genes that are identified as putative biomarkers for genotoxicity are selected based on strong transcriptional activation upon exposure to genotoxic agents.
  • the promoter regions of these genes are fused to a fluorescent reporter gene and stable integrated in mouse ES cells. Ideally, the changes in DsRed expression upon genotoxic stress would closely resemble the transcriptional activation of the endogenous biomarker genes. Expression levels of the reporter genes and the endogenous genes are determined at different times after exposure to a genotoxic agent by quantitative RT-PCR.
  • the Bscl2-GFP reporter is selectively activated after exposure to DNA damaging agents. Induction of the reporter is associated with inhibition of DNA replication and activation of the ATR DNA damage signaling pathway.
  • the Srxn1-GFP reporter is specifically induced upon oxidative stress and is part of the Nrf2 antioxidant response pathway. Employment of these mES reporter cell lines can provide insight into the primary reactive properties of known and unknown chemicals.
  • FIG. 16 shows GFP fluorescence-based mES reporter cell systems can be used to detect genotoxicity and oxidative stress.
  • FIG. 17 demonstrates that GFP reporters are specific for genotoxic and oxidative stress.
  • FIG. 18 shows Srxn1-GFP induction in response to ROS production.
  • FIG. 19 shows Bscl2-GFP reporter activation by DNA replication inhibition.
  • FIG. 20 shows that DNA damage reporter activation depends on ATR signalling.
  • FIG. 21 focuses on the interaction between p53 and Nrf2 signalling pathways and GFP reporter activation.
  • Mouse embryonic stem cell systems can detect genotoxic or oxidative stress-inducing properties of chemicals.
  • Bscl2-GFP reporter for DNA damage is activated upon DNA replication inhibition
  • Bscl2-GFP reporter expression depends on the ATR damage signalling pathway but is p53 independent.
  • Srxn1-GFP reporter for oxidative stress is activated in response to ROS production and is controlled by the Nrf2 pathway.
  • the ToxTracker Assay GFP Reporter Systems That Provide Mechanistic Insight Into the Genotoxic Properties of Chemicals
  • the ToxTracker consisting of different mES reporter cell lines that are preferentially responsive to genotoxic compounds or to agents that induce oxidative stress.
  • the Bscl2-GFP genotoxicity reporter is activated upon replication inhibition and depends on the ATR-Chk1 signaling pathway. However, Bscl2-GFP expression is not regulated by the p53 tumor suppressor.
  • the Srxn1-GFP reporter is activated upon increased levels of oxidative stress and is controlled by the Nrf2 anti-oxidant pathway.
  • a third GFP reporter cell line based on the p53-responsive Btg2 gene is activated upon exposure to a broad spectrum of (geno)toxic compounds.
  • the ToxTracker assay provides a powerful tool for (geno)toxic risk assessment of novel chemicals while providing mechanistic information on the genotoxic and/or oxidative properties of a compound.
  • C57/B16 B4418 wild type mouse ES (mES) cells were cultured in ES knockout medium (Gibco) containing 10% FCS, 2 mM glutamax, 1 mM sodium pyruvate, 100 ⁇ M ⁇ -mercaptoethanol and leukemia inhibitory factor (LIF) as previously described (Hendriks et al, 2011).
  • Mouse ES cells were propagated on irradiated primary mouse embryonic fibroblasts as feeders according to established protocols. Cells were seeded 24 h prior to chemical exposure on gelatin-coated plates in BRL-conditioned ES cell medium in the absence of feeder cells.
  • cells were exposed for 3 h in the presence of 1% S9 rat liver extract in 3.2 mM KCl, 0.8 mM MgCl 2 , 0.5 mM glucose-6-phosphate and 0.4 mM NADP. After 3 h cells were washed with PBS and cultured for 24 h in BRL-conditioned medium without the tested compounds. In all other treatments cells were continuously exposed for 24 h before GFP reporter analysis. For the inhibition of ATM, ATR and Chk1/Chk2 signaling in response to replication stress, cells were seeded 24 h prior to exposure in gelatin-coated 96-wells plates.
  • Cells were exposed to 10 ⁇ M cisplatin (CisPt) or 1.5 ⁇ M aphidicolin (Aph) for 24 h in the presence of Ku55933 ATM inhibitor (0, 2, 5, 10 ⁇ M), schisandrin B ATR inhibitor (0, 6, 15, 30 ⁇ M) or UCN-01 Chk1/Chk2 inhibitor (0, 100, 200 nM).
  • the GFP reporters were generated by BAC recombineering as described above (Poser et al, 2008).
  • Bacterial strains with a BAC containing the biomarker gene were selected using mouse BAC finder and ordered from BACPAC.
  • the putative biomarker genes on the BAC were modified with a C-terminal GFP green fluorescent marker (Poser et al, 2008) using the Quick & Easy BAC modification Kit (Gene Bridges).
  • Electrocompetent bacterial BAC strains were first transformed with the pRed/ET plasmid that contains the RecE and RecT recombination enzymes.
  • PCR fragments encoding a GFP-ires-neomycin/kanamycin reporter cassette were generated using primers that each contain 50 nucleotide additional sequence homologous to the 3′ sequence of the biomarker gene on the BAC. These homologous sequences on both the 5′ and 3′ ends of the PCR fragment allow RecE/T-mediated site-specific recombination of the GFP-ires-Neo selection cassette at the 3′ end of the biomarker gene on the BAC.
  • BAC strains that contain pRed/ET were grown at 37° C. for 30 minutes in the presence of L-arabinose to induce expression of the recombination enzymes.
  • BAC strains were transformed with the GFP-ires-Neo PCR fragment by electroporation, incubated at 37° C. for 2 h to allow recombination of the PCR fragment with the BAC and plated on kanamycin selection plates. Individual clones were analyzed for proper integration of the GFP cassette by PCR. Modified BACs were isolated using the Nucleobond PC100 DNA isolation kit (Macherey Nagel).
  • Mouse ES cells were seeded on gelatin-coated culture dishes 24 h prior to transfection. Modified BACs were transfected into the mES cells using Lipofectamine 2000 (Invitrogen) according as described previously (Poser et al, 2008). Monoclonal mES cell lines were selected based on the level of induction of the GFP reporter after exposure to genotoxic compounds or pro-oxidants. GFP expression was determined by flow cytometry.
  • the GFP reporter cells were transfected with SMARTpools of four individual siRNAs against Nrf2 or p53 (Dharmacon). A scrambled non-targeting siRNA pool was used as negative control. A siRNA against kif11, an essential gene that encodes a kinesin-like protein, was used to determine transfection efficiency. Kif11 knockdown is lethal for mES cells.
  • siRNA transfections were performed in gelatin-coated 96-wells cell culture plates. 1 ⁇ M siRNA was mixed with 0.1 ⁇ l Dharmafect 1 transfection reagent in 20 ⁇ l serum-free medium per transfection. The siRNA mix was transferred to the 96-wells plate and subsequently 11.000 mES reporter cells were seeded in each well.
  • GFP reporter expression in mES cells was visualized by confocal immunofluorescence microscopy and live cell imaging.
  • Cells were seeded at low density on fibronectin-coated glass cover slips.
  • confocal microscopy analysis the cells were exposed to 5 ⁇ M CisPt or 150 ⁇ M DEM for 24 h and subsequently fixed with 2% paraformaldehyde in PBS.
  • GFP reporter expression was visualized using a Leica TCS SP2 confocal microscope.
  • live cell imaging cells were plated on glass bottom 96 well culture plates (Greiner) and exposed to 5 ⁇ M CisPt or 100 ⁇ M DEM.
  • GFP reporter activation was determined using a Nikon TiE2000 microscope equipped with a Perfect Focus System and an automated microscope stage at 37° C. with 5% CO 2 delivery to the sample plate location. Images were acquired with a 20 ⁇ (NA 0.75) dry Plan Apochromat objective and the image acquisition was controlled by EZ-C1 software (Nikon). In each well, an image from the same position was acquired every 15 minutes for a period of 24 hours. Automated image analysis of individual images was performed using Image-Pro Plus software (MediaCybernetics) to calculate the induction of overall cellular fluorescence.
  • Image-Pro Plus software MediaCybernetics
  • Induction of the GFP reporters was compared with the expression of the endogenous gene using quantitative real time PCR (qRT-PCR).
  • Cells were exposed to the genotoxic agent CisPt or the pro-oxidant DEM and total RNA was isolated after 8 or 16 hours using the RNeasy mini kit (Qiagen).
  • cDNA was synthesized using oligo(dT) 12-18 primers and SuperscriptIII reverse transcriptase (Invitrogen) according to the manufacturer's protocol.
  • Expression of the GFP reporter and the endogenous biomarker gene was determined using specific primers against the 3′-UTR of either gene with the FastStart SYBR Green Master (Roche) QPCR mix on a 7900HT Fast Real-Time PCR System (Applied Biosystems). Relative expression was normalized using expression of the YWHAD and Hprt genes.
  • Activation of the ATM and ATR signaling pathways in response to CisPt and Aph was determined by Western blot analysis.
  • Cells were lysed in Laemmli protein sample buffer after 24 h exposure and subjected to SDS-PAGE. Proteins were transferred to PVDF membrane (Millipore) and detected using antibodies against phospho-Kap1 (Bethyl laboratories), phospho-Chk1 (Bethyl laboratories), phospho-p53 (Cell signaling) or Hprt (Santa Cruz) as protein loading control. Proteins were visualized using enhanced chemoluminescence (ECL).
  • ECL enhanced chemoluminescence
  • the mES GFP reporter cells were exposed to at least five different concentrations of 50 genotoxic and non-genotoxic compounds.
  • the selection of compounds was largely based on the ECVAM suggested list of chemicals for validation of in vitro genotoxicity test assays (Kirkland et al, 2008).
  • Compound concentrations that were used for the validation were based on cytotoxicity, where the highest concentration induced significant cell death (10-25% viable cells after 24 h treatment).
  • Cell viability was determined by flow cytometry as the fraction of intact cells after 24 h of treatment compared to untreated cells. For compounds that did not affect cell viability, a maximum concentration of 10 mM was used. Induction of GFP fluorescence in the reporter cells was determined after 24 h exposure by flow cytometry.
  • Activation of a reporter cell line was considered positive when exposure to at least two different concentrations of a compound resulted in >1.5 fold induction of GFP expression which is at least 5 times higher than the standard deviation in background fluorescence.
  • concentrations that resulted in >25% cell survival were taken into account. At higher cell killing levels GFP induction would often not increase anymore with dose. All presented data are the summary of at least three independent experiments. All shown error bars represent standard deviations.
  • the Bscl2 gene was shown to be selectively responsive to genotoxic compounds.
  • the Bscl2 gene is defective in patients suffering from Berardinelli-Seip congenital lipodystrophy and encodes the Seipin protein (Magre et al, 2001)(Szymanski et al, 2007). So far, the Bscl2 gene has not been implicated in the cellular DNA damage response.
  • the Srxn1 gene encodes the sulfiredoxin-1 protein that reduces oxidized cysteines in peroxiredoxins (Prxs) in the peroxisomes. Srxn1 plays an important role in the defense against cellular oxidative stress (Chang et al, 2004).
  • BAC recombineering Pieris et al, 2008
  • FIG. 16B To allow physiological regulation of gene expression we used BAC recombineering (Poser et al, 2008) ( FIG. 16B ) to generate Bscl2- and Srxn1-based green fluorescent reporters. Following transfection multiple mES cell lines containing either the Bscl2-GFP or Srxn1-GFP reporter were evaluated for their responsiveness to either the DNA damaging agent cisplatin (CisPt) or the indirect pro-oxidant diethyl maleate (DEM) and a single clonal cell line for either construct was selected for further studies.
  • CisPt DNA damaging agent
  • DEM indirect pro-oxidant diethyl maleate
  • Bscl2-GFP correctly localized to the endoplasmic reticulum, while Srxn1-GFP was located in both the nucleus and cytoplasm as reported previously, indicating that the GFP tag did not affect their localization ( FIG. 16C ).
  • qRT-PCR quantitative real-time PCR
  • the Srxn1-GFP reporter was somewhat responsive to CisPt but highly induced after exposure to DEM.
  • the specificity of the Srxn1-GFP reporter was comparable to endogenous Srxn1 although the extent of induction of the GFP reporter appears to be slightly higher compared to the endogenous gene.
  • MMS is a DNA alkylating agent
  • we and others have previously shown that the primary toxic response of cells after exposure to MMS is strongly correlated with oxidative stress induction, likely due to the direct reaction of MMS with gluthatione and other proteins (Hendriks et al, 2011)(Wilhelm et al, 1997)(Ashino et al, 2003).
  • the Bscl2-GFP reporter was significantly induced after exposure to all tested genotoxic compounds in a concentration-dependent fashion but hardly responded to either of the pro-oxidants or the alkylating agent MMS ( FIG. 17D ).
  • the Srxn1-GFP reporter cells was highly responsive to all oxidative stress-inducing agents including MMS, but was also induced by the genotoxic compounds, albeit to a lesser extent than the Bscl2-GFP reporter. Cytotoxicity of CisPt and DEM was comparable in the Bscl2-GFP and Srxn1-GFP reporter cells, indicating that induction of the GFP reporters is not correlated with general cellular stress ( FIG. 17B ). In addition, both GFP reporter cell lines were equally sensitive to CisPt and DEM indicating that expression of the GFP reporters is not cytotoxic.
  • FIG. 17E We evaluated the dynamic response of the GFP reporter cell lines by using time-lapse live cell imaging confocal microscopy and quantitative image analysis.
  • the Srxn1-GFP reporter was readily induced upon exposure to DEM but was hardly responsive to CisPt along the entire time period. Expression of the Srxn1-GFP reporter was clearly detectable after 8 h exposure to DEM and reached a plateau after 24 h exposure. Reversely, the Bscl2-GFP reporter was preferentially induced upon exposure to CisPt, in agreement with the flow cytometry analysis ( FIG. 17D ). Bscl2-GFP expression became visible as early as 12 h and steadily increased up to 24 h after start of treatment.
  • ECVAM Class 1 compounds consists of in vivo carcinogens that are either positive or negative in the Ames bacterial mutagenicity test or that should score positive in an in vitro genotoxicity assay.
  • ECVAM Class 2 compounds are Ames negative, non-genotoxic carcinogens or non-carcinogens that should be negative in in vitro genotoxicity tests and ECVAM Class 3 compounds are Ames negative but score equivocal or positive in an in vitro genotoxicity assay.
  • ECVAM-suggested genotoxins pro-oxidants and non-genotoxins were performed (Table 3 and FIGS. 22-25 ). All ECVAM class 1 compounds scored positive in one or both GFP reporter cell lines, except p-chloroaniline.
  • Tert-butylhydroquinone 1948-33-0 Neg. Neg. Pos. Pos. Pos. Pro-oxidant o-anthranilic acid 118-92-3 Neg. Neg. Pos. Pos. Neg. 1,3-Dihydroxybenzene 108-46-3
  • Genotoxin DNA crosslinking agent Used in cancer treatment. Etoposide 33419-42-0 Neg. Pos. Pos. Pos. Pos. Genotoxin Topoisomerase II poison. Diethyl maleate 141-05-9 nd nd nd Pos. Pos. Pro-oxidant Pro-oxidant. Tert-butyl 75-91-2 Pos. Neg. Neg. Neg. Pos. Pro-oxidant Pro-oxidant. hydroperoxide Hydrogen peroxide 7722-84-1 Pos. Neg. Pos. Neg. Pos. Pro-oxidant Pro-oxidant. Flavopiridol 146426-40-6 nd nd nd Pos. Neg. Cdk2 inhibitor. Cell cycle blocking agent. Copper sulfate 7758-98-7 nd nd nd Pos.
  • Pro-oxidant Used as herbicide, fungicide and pesticide. Potassium bromate 7758-01-2 Pos. Pos. Pos. nd Pos. Pro-oxidant Pro-oxidant. Used as flour additive. 4-Nitroquinelone- 56-57-5 Pos. Pos. Pos. nd Pos. Genotoxin UV-mimetic agent. 1-oxide 4-Hydroxy-2-nonenal 75899-68-2 Neg. Neg. Pos. nd Pos. Pro-oxidant Lipid peroxi- dation product. Cytarabine 147-94-4 Neg. nd Pos. nd Pos. Genotoxin DNA chain terminator. Used in chemotherapy. Camptothecin 7689-03-4 Neg. Neg. Pos. nd Pos.
  • Nrf2 is a key regulator of the induced expression of anti-oxidative enzymes in response to oxidative stress (Hayes and McLellan, 1999). Also Srxn1 has previously been identified as a potential Nrf2 target gene (Singh et al, 2009). To investigate whether the Nrf2 pathway controls expression of the Srxn1-GFP reporter, we exposed reporter cells to various genotoxic and oxidative stress-inducing agents following siRNA mediated knock down of Nrf2 ( FIG. 27B ).
  • Srxn1-GFP was preferentially induced by the pro-oxidants DEM, MMS and NaAsO 2 while the Bscl2-GFP reporter was selectively activated by the genotoxic agents CisPt and etoposide.
  • the response of both GFP reporter cell lines to genotoxic agents was not affected by Nrf2 knockdown.
  • induction of the Srxn1-GFP reporter by pro-oxidants was strongly decreased after knockdown of Nrf2, indicating that Srxn1-GFP is under control of the Nrf2 anti-oxidant response ( FIG. 27C ).
  • Bscl2-GFP Reporter is Activated by DNA Replication Stress
  • the Bscl2-GFP reporter is activated by a wide variety of genotoxic chemical compounds with different reactive properties ( FIGS. 16A and 17E and Table 3).
  • CisPt and MMC are DNA crosslinking agents, etoposide and doxorubicin induce DNA double strand breaks during DNA replication by inhibition of topoisomerase II, while MNU methylates DNA.
  • MNU methylates DNA.
  • a common property of genotoxic compounds is that they cause DNA damage that can interfere with transcription and DNA replication.
  • HU Hydroxyurea
  • ribonucleotide reductase ribonucleotide reductase
  • Aph aphidicolin
  • Both drugs inhibit DNA replication without inflicting DNA damage.
  • Exposure of Bscl2-GFP cells to HU or Aph resulted in a significant induction of GFP expression, indicating that activation of the Bscl2-GFP reporter by various genotoxic agents is related to DNA replication stress ( FIG. 28A ).
  • Exposure of Srxn1-GFP cells to HU or Aph resulted in slight reporter activation, in agreement with its limited activation by various genotoxic agents ( FIGS. 28A and 17E ).
  • Bscl2-GFP reporter activation could almost completely be repressed by either the ATR or the Chk1/Chk2 inhibitor, but was unaffected by the ATM inhibitor ( FIG. 28C ). These data indicate that the Bscl2-GFP reporter is activated by the ATR-Chk1 signaling pathway in response to stalled DNA replication forks. As previously, while the Srxn1-GFP reporter is also slightly activated upon exposure to CisPt or Aph, its activation is not dependent on the ATM or ATR DNA damage signaling pathways.
  • the p53 tumor suppressor plays a central role in the DNA damage response (Meek, 2009).
  • p53 is activated by various cellular signaling pathways, including the ATM-Chk2 and ATR-Chk1 kinases, (see also FIG. 28B ), and controls the activity of DNA repair systems, cell cycle checkpoints and apoptosis.
  • induction of the Nrf2 pathway also appears to trigger p53 activation (Wakabayashi et al, 2010).
  • p53 also affects expression of Nrf2 target genes (Wakabayashi et al, 2010).
  • FIG. 29A To investigate whether the Bscl2-GFP and Srxn1-GFP reporters were under control of p53, we transiently knocked down p53 expression by siRNAs ( FIG. 29A ), exposed cells to various genotoxic and oxidative stress-inducing compounds and analyzed GFP reporter activation. Activation of neither the Bscl2-GFP nor the Srxn1-GFP reporter by any of the tested compounds was affected by p53 knockdown ( FIG. 29B ). To confirm that the extent of p53 knockdown was sufficient to prevent activation of p53 target genes, we employed a Btg2-GFP reporter.
  • Btg2 is a known p53 target gene that is transcriptionally activated upon exposure to genotoxic and oxidative stress (Rouault et al, 1996). Exposure of Btg2-GFP reporter cells to genotoxic agents as well as pro-oxidants resulted in increased expression of the reporter ( FIG. 29C ). Knockdown of p53 resulted in a strongly reduced induction of the Btg2-GFP reporter after exposure to all tested compounds. Interestingly, also knockdown of Nrf2 resulted in decreased Btg2-GFP reporter activation, specifically after exposure to oxidative stress-inducing compounds.
  • ToxTracker assay which consists of different GFP fluorescence mES reporter cell lines that are preferentially responsive to either genotoxic or oxidative stress-inducing compounds.
  • Expression of the Bscl2-GFP reporter is specifically induced when mES cells are exposed to genotoxic compounds but is not elevated by chemicals that induce oxidative stress even though oxygen radicals might also damage DNA ( FIG. 17 ).
  • Bscl2-GFP reporter activation correlates with inhibition of DNA replication progression ( FIG. 28 ).
  • Most types of oxidative DNA lesions do not provide strong blocks for the DNA replication machinery and are therefore unlikely to induce expression of the Bscl2-GFP reporter (Tolentino et al, 2008).
  • Srxn1-GFP reporter is also somewhat responsive to genotoxic compounds. This suggests that either Srxn1 gene expression is directly induced upon DNA damage, although expression of Srxn1 is independent of the ATR-Chk1 DNA damage signaling ( FIG. 28C ), or that exposure of cells to genotoxic agents also results in increased ROS levels. Indeed, exposure of cells to the DNA crosslinking agent CisPt was shown to result in increased levels of ROS, caused by impaired mitochondrial function and during apoptosis (Jing et al, 2007).
  • the Bscl2 gene has originally been identified in patients that suffer from Berardinelli-Seip congenital lipodystrophy, a rare autosomal recessive disease that is characterized by an almost complete absence of adipocytes (Magre et al, 2001).
  • the Bscl2 gene encodes a protein called Seipin that is located to the membrane of the endoplasmic reticulum (Szymanski et al, 2007).
  • mutations in the Bscl2 gene have also been associated with autosomal-dominant disorders of motoneurons that results in severe atrophy and wasting of distal limb muscles (Agarwal and Garg, 2004).
  • Seipin is involved in adipocyte differentiation, it is mainly expressed in the nervous system and testis (Magre et al, 2001). The function of Seipin is largely unknown, although it has been implicated in cytosolic lipid droplet morphology and in intracellular transport of lipids and proteins. So far, there is no implication of Bscl2 in the DNA damage response. Our data show that expression of Bscl2 in mES cells is strongly induced upon exposure to various DNA damaging agents. Basal Bscl2 expression level is low in mES cells.
  • Bscl2 expression has been associated with adipocyte differentiation, it is attractive to hypothesize that also in mES cells Bscl2 expression is correlated with the induction of cell differentiation after exposure to genotoxic agents. It is well established that cell differentiation is induced upon DNA damage to maintain genome stability and to prevent malignant transformation of stem cells (Sherman et al, 2011). In agreement, exposure to various genotoxic chemicals did not result in increased expression of the Bscl2 gene in primary liver cells (M. Schaap, personal communication) and only a marginal induction in HepG2 liver carcinoma cells (Magkoufopoulou et al. Submitted). We are currently investigating the role of Bscl2 in the DNA damage response in mES cells.
  • the Bscl2-GFP reporter is exclusively induced by compounds that affect ongoing DNA replication, while the Srxn1-GFP reporter is preferentially induced by oxidative stress.
  • the ToxTracker assay that consists of three independent GFP-based reporter cell lines is a novel, highly sensitive and specific genotoxicity test that can provide insights in the relative toxicity potential of chemicals.

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